Improvement of Carotenoids' Production by Increasing the Activity of Beta-Carotene Ketolase with Different Strategies.

Canthaxanthin is an important antioxidant with wide application prospects, and ß-carotene ketolase is the key enzyme involved in the biosynthesis of canthaxanthin. However, the challenge for the soluble expression of ß-carotene ketolase is that it hinders the large-scale production of carotenoids such as canthaxanthin and astaxanthin. Hence, this study employed several strategies aiming to improve the soluble expression of ß-carotene ketolase and its activity, including selecting optimal expression vectors, screening induction temperatures, adding soluble expression tags, and adding a molecular chaperone. Results showed that all these strategies can improve the soluble expression and activity of ß-carotene ketolase in Escherichia coli. In particular, the production of soluble ß-carotene ketolase was increased 8 times, with a commercial molecular chaperon of pG-KJE8, leading to a 1.16-fold enhancement in the canthaxanthin production from ß-carotene. Interestingly, pG-KJE8 could also enhance the soluble expression of ß-carotene ketolase derived from eukaryotic microalgae. Further research showed that the production of canthaxanthin and echinenone was significantly improved by as many as 30.77 times when the pG-KJE8 was added, indicating the molecular chaperone performed differently among different ß-carotene ketolase. This study not only laid a foundation for further research on the improvement of ß-carotene ketolase activity but also provided new ideas for the improvement of carotenoid production.

A chromosome-level genome assembly for the astaxanthin-producing microalga Haematococcus pluvialis.

The green microalga Haematococcus pluvialis can synthesize high amounts of astaxanthin, which is a valuable antioxidant that has been utilized in human health, cosmetics, and aquaculture. To illustrate detailed molecular clues to astaxanthin yield, we performed PacBio HIFI along with Hi-C sequencing to construct an improved chromosome-level haplotypic genome assembly with 32 chromosomes and a genome size of 316.0 Mb. Its scaffold N50 (942.6 kb) and contig N50 (304.8 kb) have been upgraded remarkably from our previous genome draft, and a total of 32,416 protein-coding genes were predicted. We also established a high-evidence phylogenetic tree from seven representative algae species, with the main aim to calculate their divergence times and identify expanded/contracted gene families. We also characterized genome-wide localizations on chromosomes of some important genes such as five BKTs (encoding beta-carotene ketolases) that are putatively involved in astaxanthin production. In summary, we reported the first chromosome-scale map of H. pluvialis, which provides a valuable genetic resource for in-depth biomedical investigations on this momentous green alga and commercial astaxanthin bioproduction.

Enhancement of ß-carotene content in Chlamydomonas reinhardtii by expressing bacterium-driven lycopene ß-cyclase.

ß-Carotene is one of the economically important carotenoids, having functions as the antioxidant to remove harmful free radicals and as the precursor for vitamin A and other high-valued xanthophyll such as zeaxanthin and astaxanthin. Lycopene cyclase plays an important role in the branching of ß-carotene and α-carotene. Aiming to develop the microalgae with enhanced ß-carotene productivity, the CrtY gene from bacterium Pantoea agglomerans was integrated into Chlamydomonas reinhardtii. The lycopene-producing E. coli harboring CrtY gene produced 1.59 times of ß-carotene than that harboring DsLcyb1 from Dunaliella salina (a microalga with abundant ß-carotene), confirming the superior activity of CrtY on ß-carotene biosynthesis. According to the pigment analysis by HPLC, in microalgal transformants that were confirmed by molecular analysis, the expression of CrtY significantly increased ß-carotene content from 12.48 mg/g to 30.65 mg/g (dry weight), which is about 2.45-fold changes. It is noted that three out of five transformants have statistically significant higher amount of lutein, even though the increment was 20% in maximum. Besides, no growth defect was observed in the transformants. This is the first report of functional expression of prokaryotic gene in eukaryotic microalgae, which will widen the gene pool targeting carotenoids biosynthesis using microalgae as the factory and thereby provide more opportunity for high-valued products engineering in microalgae.

Comparing the Ability of Secretory Signal Peptides for Heterologous Expression of Anti-Lipopolysaccharide Factor 3 in Chlamydomonas reinhardtii.

Anti-lipopolysaccharide factor 3 (ALFPm3) possesses a wide antimicrobial spectrum and high antibacterial and viral activities for broad application prospects in the aquaculture industry. However, the application of ALFPm3 is limited by its low production in nature, as well as its low activity when expressed in Escherichia coli and yeast. Although it has been proven that its secretory expression can be used to produce antimicrobial peptides with strong antimicrobial activity, there is no study on the high-efficiency secretory expression of ALFPm3 in Chlamydomonas reinhardtii. In this study, signal peptides ARS1 and CAH1 were fused with ALFPm3 and inserted into the pESVH vector to construct pH-aALF and pH-cALF plasmids, respectively, that were transformed to C. reinhardtii JUV using the glass bead method. Subsequently, through antibiotic screening, DNA-PCR, and RT-PCR, transformants expressing ALFPm3 were confirmed and named T-JaA and T-JcA, respectively. The peptide ALFPm3 could be detected in algal cells and culture medium by immunoblot, meaning that ALFPm3 was successfully expressed in C. reinhardtii and secreted into the extracellular environment. Moreover, ALFPm3 extracts from the culture media of T-JaA and T-JcA showed significant inhibitory effects on the growth of V. harveyi, V. alginolyticus, V. anguillarum, and V. parahaemolyticus within 24 h. Interestingly, the inhibitory rate of c-ALFPm3 from T-JcA against four Vibrio was 2.77 to 6.23 times greater than that of a-ALFPm3 from T-JaA, indicating that the CAH1 signal peptide was more helpful in enhancing the secreted expression of the ALFPm3 peptide. Our results provided a new strategy for the secretory production of ALFPm3 with high antibacterial activity in C. reinhardtii, which could improve the application potentiality of ALFPm3 in the aquaculture industry.

Secretory Expression and Application of Antilipopolysaccharide Factor 3 in Chlamydomonas reinhardtii.

Anti-lipopolysaccharide factor is a class of antimicrobial peptides with lipopolysaccharide-binding structural domains, which has a broad antimicrobial spectrum, high antimicrobial activities, and broad application prospects in terms of the aquaculture industry. However, the low yield of natural antimicrobial peptides and their poor expression activity in bacteria and yeast have hindered their exploration and utilization. Therefore, in this study, the extracellular expression system of Chlamydomonas reinhardtii, by fusing the target gene with the signal peptide, was used to express anti-lipopolysaccharide factor 3 (ALFPm3) from Penaeus monodon in order to obtain highly active ALFPm3. Transgenic C. reinhardtii T-JiA2, T-JiA3, T-JiA5, and T-JiA6, were verified using DNA-PCR, RT-PCR, and immunoblot. Additionally, the IBP1-ALFPm3 fusion protein could be detected not only within the cells but also in the culture supernatant. Moreover, the extracellular secretion containing ALFPm3 was collected from algal cultures, and then its bacterial inhibitory activity was analyzed. The results showed that the extracts from T-JiA3 had an inhibition rate of 97% against four common aquaculture pathogenic bacteria, including Vibrio harveyi, Vibrio anguillarum, Vibrio alginolyticus, and Vibrio parahaemolyticus. The highest inhibition rate of 116.18% was observed in the test against V. anguillarum. Finally, the minimum inhibition concentration (MIC) of the extracts from T-JiA3 to V. harveyi, V. anguillarum, V. alginolyticus, and V. parahaemolyticus were 0.11 µg/µL, 0.088 µg/µL, 0.11 µg/µL, and 0.011 µg/µL, respectively. This study supports the foundation of the expression of highly active anti-lipopolysaccharide factors using the extracellular expression system in C. reinhardtii, providing new ideas for the expression of highly active antimicrobial peptides.

Research Progress in Heterologous Crocin Production.

Crocin is one of the most valuable components of the Chinese medicinal plant Crocus sativus and is widely used in the food, cosmetics, and pharmaceutical industries. Traditional planting of C. sativus is unable to fulfill the increasing demand for crocin in the global market, however, such that researchers have turned their attention to the heterologous production of crocin in a variety of hosts. At present, there are reports of successful heterologous production of crocin in Escherichia coli, Saccharomyces cerevisiae, microalgae, and plants that do not naturally produce crocin. Of these, the microalga Dunaliella salina, which produces high levels of ß-carotene, the substrate for crocin biosynthesis, is worthy of attention. This article describes the biosynthesis of crocin, compares the features of each heterologous host, and clarifies the requirements for efficient production of crocin in microalgae.

The Histone Acetyltransferase HpGCN5 Involved in the Regulation of Abiotic Stress Responses and Astaxanthin Accumulation in Haematococcus pluvialis.

The histone acetyltransferases (HATs), together with histone deacetylases, regulate the gene transcription related to various biological processes, including stress responses in eukaryotes. This study found a member of HATs (HpGCN5) from a transcriptome of the economically important microalgae Haematococcus pluvialis. Its expression pattern responding to multiple abiotic stresses and its correlation with transcription factors and genes involved in triacylglycerols and astaxanthin biosynthesis under stress conditions were evaluated, aiming to discover its potential biological function. The isolated HpGCN5 was 1,712 bp in length encoding 415 amino acids. The signature domains of Acetyltransf_1 and BROMO were presented, as the GCN5 gene from Arabidopsis and Saccharomyces cerevisiae, confirming that HpGCN5 belongs to the GCN5 subfamily of the GNAT superfamily. The phylogenetic analysis revealed that HpGCN5 is grouped with GNAT genes from algae and is closer to that from higher plants, compared with yeast, animal, fungus, and bacteria. It was predicted that HpGCN5 is composed of 10 exons and contains multiple stress-related cis-elements in the promoter region, revealing its potential role in stress regulation. Real-time quantitative PCR revealed that HpGCN5 responds to high light and high salt stresses in similar behavior, evidenced by their down-regulation exposing to stresses. Differently, HpGCN5 expression was significantly induced by SA and Nitrogen-depletion stresses at the early stage but was dropped back after then. The correlation network analysis suggested that HpGCN5 has a strong correlation with major genes and a transcription factor involved in astaxanthin biosynthesis. Besides, the correlation was only found between HpGCN5 and a few genes involved in triacylglycerols biosynthesis. Therefore, this study proposed that HpGCN5 might play a role in the regulation of astaxanthin biosynthesis. This study firstly examined the role of HATs in stress regulation and results will enrich our understanding of the role of HATs in microalgae.

Chemical synthesis of left arm of Chlamydomonas reinhardtii mitochondrial genome and in vivo functional analysis.

Chlamydomonas reinhardtii is a photosynthetic eukaryote showing great industrial potential. The synthesis and in vivo function of the artificial C. reinhardtii genome not only promotes the development of synthetic biology technology but also supports industries that utilize this algae. Mitochondrial genome (MtG) is the smallest and simplest genome of C. reinhardtii that suits synthetic exploration. In this article, we designed and assembled a synthetic mitochondria left arm (syn-LA) genome sharing >92% similarity to the original mitochondria genome (OMtG) left arm, transferred it into the respiratory defect strain cc-2654, screened syn-LA containing transformants from recovered dark-growth defects using PCR amplification, verified internal function of syn-LA via western blot, detected heteroplasmic ratio of syn-LA, tried promoting syn-LA into homoplasmic status with paromomycin stress, and discussed the main limitations and potential solutions for this area of research. This research supports the functionalization of a synthetic mitochondrial genome in living cells. Although further research is needed, this article nevertheless provides valuable guidance for the synthesis of eukaryotic organelle genomes and opens possible directions for future research.

Haematococcus pluvialis Accumulated Lipid and Astaxanthin in a Moderate and Sustainable Way by the Self-Protection Mechanism of Salicylic Acid Under Sodium Acetate Stress.

To elucidate the mechanism underlying increased fatty acid and astaxanthin accumulation in Haematococcus pluvialis, transcriptome analysis was performed to gain insights into the multiple defensive systems elicited by salicylic acid combined with sodium acetate (SAHS) stresses with a time course. Totally, 112,886 unigenes and 61,323 non-repeat genes were identified, and genes involved in carbon metabolism, primary and secondary metabolism, and immune system responses were identified. The results revealed that SA and NaAC provide both energy and precursors to improve cell growth of H. pluvialis and enhance carbon assimilation, astaxanthin, and fatty acids production in this microalga with an effective mechanism. Interestingly, SA was considered to play an important role in lowering transcriptional activity of the fatty acid and astaxanthin biosynthesis genes through self-protection metabolism in H. pluvialis, leading to its adaption to HS stress and finally avoiding massive cell death. Moreover, positive correlations between 15 key genes involved in astaxanthin and fatty acid biosynthesis pathways were found, revealing cooperative relation between these pathways at the transcription level. These results not only enriched our knowledge of the astaxanthin accumulation mechanism in H. pluvialis but also provided a new view on increasing astaxanthin production in H. pluvialis by a moderate and sustainable way in the future.

Transcription Factors From Haematococcus pluvialis Involved in the Regulation of Astaxanthin Biosynthesis Under High Light-Sodium Acetate Stress.

The microalgae Haematococcus pluvialis attracts attention for its ability to accumulate astaxanthin up to its 4% dry weight under stress conditions, such as high light, salt stress, and nitrogen starvation. Previous researches indicated that the regulation of astaxanthin synthesis might happen at the transcriptional level. However, the transcription regulatory mechanism of astaxanthin synthesis is still unknown in H. pluvialis. Lacking studies on transcription factors (TFs) further hindered from discovering this mechanism. Hence, the transcriptome analysis of H. pluvialis under the high light-sodium acetate stress for 1.5 h was performed in this study, aiming to discover TFs and the regulation on astaxanthin synthesis. In total, 83,869 unigenes were obtained and annotated based on seven databases, including NR, NT, Kyoto Encyclopedia of Genes and Genomes Orthology, SwissProt, Pfam, Eukaryotic Orthologous Groups, and Gene Ontology. Moreover, 476 TFs belonging to 52 families were annotated by blasting against the PlantTFDB database. By comparing with the control group, 4,367 differentially expressed genes composing of 2,050 upregulated unigenes and 2,317 downregulated unigenes were identified. Most of them were involved in metabolic process, catalytic activity, single-organism process, single-organism cellular process, and single-organism metabolic process. Among them, 28 upregulated TFs and 41 downregulated TFs belonging to 27 TF families were found. The transcription analysis showed that TFs had different transcription modules responding to the high light and sodium acetate stress. Interestingly, six TFs belonging to MYB, MYB_related, NF-YC, Nin-like, and C3H families were found to be involved in the transcription regulation of 27 astaxanthin synthesis-related genes according to the regulatory network. Moreover, these TFs might affect astaxanthin synthesis by directly regulating CrtO, showing that CrtO was the hub gene in astaxanthin synthesis. The present study provided new insight into a global view of TFs and their correlations to astaxanthin synthesis in H. pluvialis.

The Papain-like Cysteine Protease HpXBCP3 from Haematococcus pluvialis Involved in the Regulation of Growth, Salt Stress Tolerance and Chlorophyll Synthesis in Microalgae.

The papain-like cysteine proteases (PLCPs), the most important group of cysteine proteases, have been reported to participate in the regulation of growth, senescence, and abiotic stresses in plants. However, the functions of PLCPs and their roles in stress response in microalgae was rarely reported. The responses to different abiotic stresses in Haematococcus pluvialis were often observed, including growth regulation and astaxanthin accumulation. In this study, the cDNA of HpXBCP3 containing 1515 bp open reading frame (ORF) was firstly cloned from H. pluvialis by RT-PCR. The analysis of protein domains and molecular evolution showed that HpXBCP3 was closely related to AtXBCP3 from Arabidopsis. The expression pattern analysis revealed that it significantly responds to NaCl stress in H. pluvialis. Subsequently, transformants expressing HpXBCP3 in Chlamydomonas reinhardtii were obtained and subjected to transcriptomic analysis. Results showed that HpXBCP3 might affect the cell cycle regulation and DNA replication in transgenic Chlamydomonas, resulting in abnormal growth of transformants. Moreover, the expression of HpXBCP3 might increase the sensitivity to NaCl stress by regulating ubiquitin and the expression of WD40 proteins in microalgae. Furthermore, the expression of HpXBCP3 might improve chlorophyll content by up-regulating the expression of NADH-dependent glutamate synthases in C. reinhardtii. This study indicated for the first time that HpXBCP3 was involved in the regulation of cell growth, salt stress response, and chlorophyll synthesis in microalgae. Results in this study might enrich the understanding of PLCPs in microalgae and provide a novel perspective for studying the mechanism of environmental stress responses in H. pluvialis.

Transcriptome-based analysis of the effects of salicylic acid and high light on lipid and astaxanthin accumulation in Haematococcus pluvialis.

BACKGROUND: The unicellular alga Haematococcus pluvialis has achieved considerable interests for its capacity to accumulate large amounts of triacylglycerol and astaxanthin under various environmental stresses. To our knowledge, studies focusing on transcriptome research of H. pluvialis under exogenous hormones together with physical stresses are rare. In the present study, the change patterns at transcriptome level were analyzed to distinguish the multiple defensive systems of astaxanthin and fatty acid metabolism against exogenous salicylic acid and high light (SAHL) stresses. RESULTS: Based on RNA-seq data, a total of 112,463 unigenes and 61,191 genes were annotated in six databases, including NR, KEGG, Swiss-Prot, PFAM, COG and GO. Analysis of differentially expressed genes (DEGs) in KEGG identified many transcripts that associated with the biosynthesis of primary and secondary metabolites, photosynthesis, and immune system responses. Furthermore, 705 unigenes predicted as putative transcription factors (TFs) were identified, and the most abundant TFs families were likely to be associated with the biosynthesis of astaxanthin and fatty acid in H. pluvialis upon exposure to SAHL stresses. Additionally, majority of the fifteen key genes involved in astaxanthin and fatty acid biosynthesis pathways presented the same expression pattern, resulting in increased accumulation of astaxanthin and fatty acids in single celled H. pluvialis, in which astaxanthin content increased from 0.56 ± 0.05 mg·L-1 at stage Control to 0.89 ± 0.12 mg·L-1 at stage SAHL_48. And positive correlations were observed among these studied genes by Pearson Correlation (PC) analysis, indicating the coordination between astaxanthin and fatty acid biosynthesis. In addition, protein-protein interaction (PPI) network analysis also demonstrated that this coordination might be at transcriptional level. CONCLUSION: The results in this study provided valuable information to illustrate the molecular mechanisms of coordinate relations between astaxanthin and fatty acid biosynthesis. And salicylic acid might play a role in self-protection processes of cells, helping adaption of H. pluvialis to high light stress.

Morphological and Molecular Characterization of Pratylenchus dakotaensis n. sp. (Nematoda: Pratylenchidae), a New Root-Lesion Nematode Species on Soybean in North Dakota, USA.

Root-lesion nematodes (Pratylenchus spp.) of the genus Pratylenchus Filipjev, 1936, are among the most important nematode pests on soybean (Glycine max (L.) Merr.), along with soybean cyst and root-knot nematodes. In May 2015 and 2016, a total of six soil samples were collected from a soybean field in Walcott, Richland County, ND and submitted to the Mycology and Nematology Genetic Diversity and Biology Laboratory (MNGDBL), USDA, ARS, MD for analysis. Later, in 2019, additional nematodes recovered from a greenhouse culture on soybean originally from the same field were submitted for further analysis. Males, females, and juveniles of Pratylenchus sp. were recovered from soil and root samples and were examined morphologically and molecularly. DNA from single nematodes were extracted, and the nucleotides feature of three genomic regions targeting on the D2-D3 region of 28S rDNA and ITS rDNA and mitochondrial cytochrome oxidase subunit I (COX1) gene were characterized. Phylogeny trees were constructed to ascertain the relationships with other Pratylenchus spp., and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to provide a rapid and reliable differentiation from other common Pratylenchus spp. Molecular features indicated that it is a new, unnamed Pratylenchus sp. that is different from morphologically closely related Pratylenchus spp., including P. convallariae, P. pratensis, P. fallax, and P. flakkensis. In conclusion, both morphological and molecular observations indicate that the North Dakota isolate on soybean represents a new root-lesion nematode species which is named and described herein as Pratylenchus dakotaensis n. sp.

Discovery of Geranylgeranyl Pyrophosphate Synthase (GGPPS) Paralogs from Haematococcus pluvialis Based on Iso-Seq Analysis and Their Function on Astaxanthin Biosynthesis.

Haematococcus pluvialis is widely distributed in the world and well known as the richest natural source of astaxanthin that is a strong antioxidant with excellent commercial value. The pathway of astaxanthin biosynthesis in H. pluvialis has been documented as an enzymatic reaction. Several enzymes have been reported, but their isoforms or homologs have not been investigated genome-wide. To better understand the astaxanthin biosynthesis pathway in H. pluvialis, eight candidates of the geranylgeranyl pyrophosphate synthase gene (HpGGPPS) predicted from Iso-seq data were isolated in this study. The length of coding region of these candidates varied from 960 bp to 1272 bp, composing of 7-9 exons. The putative amino acids of all candidates composed the signature domain of GGPPS gene. However, the motifs in the domain region are varied, indicating different bio-functions. Phylogenetic analysis revealed eight candidates can be clustered into three groups. Only two candidates in Group1 encode the synthase participating in the astaxanthin formation. The yield of astaxanthin from these two candidates, 7.1 mg/g (DW) and 6.5 mg/g (DW) respectively, is significant higher than that from CrtE (2.4 mg/g DW), a GGPPS gene from Pantoea ananatis. This study provides a potential productive pathway for astaxanthin synthesis.

Assessment of Factors Associated with Molecular Quantification of Stubby Root Nematode Paratrichodorus allius from Field Soil DNA.

Factors relating to SYBR Green-based quantitative real-time PCR (qPCR) quantification of stubby root nematode Paratrichodorus allius using soil DNA were evaluated in this study. Soils used were loamy sand from potato fields in North Dakota and Idaho. Results showed that the largest nematode individuals (body length >720 µm) produced significant lower Cq values than the smallest individuals (<359 µm), indicating more total DNA amount in the largest nematodes. Soil pre-treatments showed that autoclaved field soil had significantly reduced DNA amount and quality. The air- or oven-dried soil yielded a lower amount of DNA with similar purity, compared with natural field soil. PCR inhibitors were detected in soil DNA substrates targeting pBluescript II SK(+)-plasmid DNA. Al(NH4)(SO4)2 treatment during DNA preparation significantly reduced the inhibitors compared with post-treatment of soil DNA with polyvinylpolypyrrolidone column. The effect of PCR inhibitors on qPCR was suppressed by bovine serum albumin. Quantification results did not significantly change when increasing the number of DNA extractions from three to six per soil sample when soil grinding and grid sampling strategies were used. Two standard curves, generated from serial dilutions of plasmid DNA containing P. allius ITS1 rDNA and soil DNA containing known nematode numbers, produced similar correlations between Cq values and amount of targets. The targets in soil DNA quantified by qPCR using either standard curve correlated well with microscopic observations using both artificially and naturally infested field soils. This is the first study for assessing various factors that may affect qPCR quantification of stubby root nematodes. Results will be useful during the setup or optimization of qPCR-based quantification of plant-parasitic nematodes from soil DNA.

Molecular and antimicrobial characterization of a group G anti-lipopolysaccharide factor (ALF) from Penaeus monodon.

Anti-lipopolysaccharide factors (ALFs) are important host-defense molecules of crustaceans. They all contain a lipopolysaccharide-binding domain (LBD) and some ALFs exhibit strong antimicrobial activity. In this research, a Group G ALF from Penaeus monodon (ALFPm11) was studied. It is an anionic peptide specifically having a cationic and highly amphipathic LBD, with five positively charged residues separated by aromatic residues. It was abundantly expressed in the hepatopancreas of P. monodon normally but the expression level in other tissues was relatively low or undetectable. However, in the shrimps challenged by Vibrio, expression of ALFPm11 could be detected in all tissues. Chemically synthesized ALFPm11-LBD displayed high inhibitory activity (minimum inhibition concentration≤ 4 µM) against various bacteria, e.g. Exiguobacterium sp. L33, Bacillus sp. T2, and Acinetobacter sp. L32. It also displayed apparent activity in the agar well diffusion assay. Furthermore, it could efficiently induce agglutination of both Gram-positive and Gram-negative bacteria and cause significant membrane permeabilization of the bacteria. As a comparative study, ALFPm11-LBD showed a better or equal antimicrobial function to ALFPm3-LBD which was reported to possess strong antimicrobial activity against Gram-positive, Gram-negative bacteria and fungi. Thus, this research found a new effective ALF in P. monodon and demonstrated its antimicrobial mechanism, suggesting its potential applications in the future.

Developing a One-Step Multiplex PCR Assay for Rapid Detection of Four Stubby-Root Nematode Species, Paratrichodorus allius, P. minor, P. porosus, and Trichodorus obtusus.

Four trichodorid species, Paratrichodorus allius, P. minor, P. porosus, and Trichodorus obtusus, were found in multiple states in the United States. Traditional diagnosis based on morphology and morphometrics is laborious and requires an experienced taxonomist. Additionally, end-point diagnosis using PCR was only available for P. allius. To increase diagnostic efficiency and reduce costs, a one-step multiplex PCR assay was developed to simultaneously identify these four species using one PCR reaction. Available sequences of 18S ribosomal DNA and internal transcribed spacer 1 (ITS1) region of these species were aligned and five primers were designed. The conserved forward primer located in the 18S region, in combination with the species-specific antisense primer in the ITS1 region, amplified a single distinctive PCR fragment for each species (421/425 bp for P. allius, 190 bp for P. minor, 513 bp for P. porosus, and 353 bp for T. obtusus). In silico analysis with 10 other trichodorid species and experimental analysis using samples with these four species, 20 other plant-parasitic and three non-plant-parasitic nematodes demonstrated high specificity with the primers designed. The multiplex PCR amplified desirable fragments using a set of artificially mixed templates containing one, two, three, or four targeted species. The reliability of multiplex PCR results was demonstrated by using nematode populations isolated from infested fields from diverse geographic regions in eight states. The multiplex PCR-based tool developed in this study for the first time provides a simple, rapid, and cost-friendly assay for accurate diagnosis of the four major trichodorid nematodes in the United States.

Molecular Characterization and Identification of Stubby Root Nematode Species From Multiple States in the United States.

Stubby root nematodes (SRN) are important plant parasites infecting many crops and widely distributed in many regions of the United States. SRN transmit Tobacco rattle virus, which causes potato corky ringspot disease, thereby having a significant economic impact on the potato industry. In 2015 to 2017, 184 soil samples and 16 nematode suspensions from North Dakota, Minnesota, Idaho, Oregon, Washington, South Carolina, North Carolina, and Florida were assayed for the presence of SRN. SRN were found in 106 soil samples with population densities of 10 to 320 SRN per 200 g of soil and in eight of the nematode suspensions. Sequencing of ribosomal DNA (rDNA) or species-specific polymerase chain reaction assays revealed the presence of four SRN species, including Paratrichodorus allius, P. minor, P. porosus, and Trichodorus obtusus. Accordingly, their rDNA sequences were characterized by analyzing D2-D3 of 28S rDNA, 18S rDNA, and internal transcribed spacer (ITS) rDNA obtained in this study and retrieved from GenBank. Both intra- and interspecies variations were higher in ITS rDNA than 18S rDNA and D2-D3 of 28S rDNA. Based on phylogenetic analysis, the four SRN species formed a monophyletic group, with P. allius more closely related to P. porosus than P. minor and T. obtusus. Indel variation of ITS2 rDNA was present in P. allius populations from the same geographic regions. This study documented the occurrence of SRN species across multiple states. The intra- and interspecies genetic diversity of rDNA in this study will provide more information for understanding the evolutionary relationships of SRN and will be valuable for future studies of SRN species identification and management.